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Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing.

Kim DS, Gusti V, Dery KJ, Gaur RK - BMC Mol. Biol. (2008)

Bottom Line: Several lines of evidence suggest that theophylline-dependent inhibition of splicing is highly specific, and thermodynamic stability of RNA-theophylline complex as well as the location of BPS within this complex affects the efficiency of splicing inhibition.Finally, we have constructed an alternative splicing model pre-mRNA substrate in which theophylline caused exon skipping both in vitro and in vivo, suggesting that a small molecule-RNA interaction can modulate alternative splicing.These findings provide the ability to control splicing pattern at will and should have important implications for basic, biotechnological, and biomedical research.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA. dskim11@gmail.com

ABSTRACT

Background: Despite tremendous progress in understanding the mechanisms of constitutive and alternative splicing, an important and widespread step along the gene expression pathway, our ability to deliberately regulate gene expression at this step remains rudimentary. The present study was performed to investigate whether a theophylline-dependent "splice switch" that sequesters the branchpoint sequence (BPS) within RNA-theophylline complex can regulate alternative splicing.

Results: We constructed a series of pre-mRNAs in which the BPS was inserted within theophylline aptamer. We show that theophylline-induced sequestering of BPS inhibits pre-mRNA splicing both in vitro and in vivo in a dose-dependent manner. Several lines of evidence suggest that theophylline-dependent inhibition of splicing is highly specific, and thermodynamic stability of RNA-theophylline complex as well as the location of BPS within this complex affects the efficiency of splicing inhibition. Finally, we have constructed an alternative splicing model pre-mRNA substrate in which theophylline caused exon skipping both in vitro and in vivo, suggesting that a small molecule-RNA interaction can modulate alternative splicing.

Conclusion: These findings provide the ability to control splicing pattern at will and should have important implications for basic, biotechnological, and biomedical research.

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Related in: MedlinePlus

Insertion of BPS within theophylline-responsive riboswitch confers ligand-dependent control of splicing. (A) Schematic diagram of AdBPT pre-mRNAs. Underlined sequence and encircled adenosine residue represents the BPS and branch nucleotide, respectively. Open boxes represent exon sequences and horizontal lines between exons indicate introns. Arrow specifies 5' and 3' splice sites. The boxed residues within the secondary structure of theophylline binding aptamer are conserved for theophylline binding. The numbering system follows according to the reference [29]. (B) In vitro splicing of AdML 21AG and AdBPT pre-mRNAs. 32P-labeled pre-mRNAs were incubated in HeLa nuclear extracts at 30°C for 2 h in the absence or presence of theophylline (for experimental details, see methods section). Total RNA isolated from each reaction was fractionated on a 13% polyacrylamide denaturing gel. The bands corresponding to pre-mRNA substrates, intermediates and spliced products are indicated on the right. M, Century™-plus RNA size marker (Ambion). Asterisk (*) indicates degraded lariat. (C) Histogram depicting the effect of theophylline on the splicing of AdML 21AG and AdBPT pre-mRNAs. Splicing was calculated as the ratio of spliced mRNA to the total and normalized to the control (no theophylline). Data represent mean ± standard error of the mean (SEM) of three independent experiments. Asterisk represents significant change as compared to the control (*, P < 0.0005). (D) Theophylline inhibits the splicing of AdBPT15AG pre-mRNA in a dose-dependent manner. 32P-labeled AdBPT15AG pre-mRNA was incubated in HeLa nuclear extracts in the absence or with increasing concentration of theophylline for 2 h as described in (B). % Splicing calculated as described above and plotted against theophylline (mM). The values are expressed as mean ± SEM.
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Figure 1: Insertion of BPS within theophylline-responsive riboswitch confers ligand-dependent control of splicing. (A) Schematic diagram of AdBPT pre-mRNAs. Underlined sequence and encircled adenosine residue represents the BPS and branch nucleotide, respectively. Open boxes represent exon sequences and horizontal lines between exons indicate introns. Arrow specifies 5' and 3' splice sites. The boxed residues within the secondary structure of theophylline binding aptamer are conserved for theophylline binding. The numbering system follows according to the reference [29]. (B) In vitro splicing of AdML 21AG and AdBPT pre-mRNAs. 32P-labeled pre-mRNAs were incubated in HeLa nuclear extracts at 30°C for 2 h in the absence or presence of theophylline (for experimental details, see methods section). Total RNA isolated from each reaction was fractionated on a 13% polyacrylamide denaturing gel. The bands corresponding to pre-mRNA substrates, intermediates and spliced products are indicated on the right. M, Century™-plus RNA size marker (Ambion). Asterisk (*) indicates degraded lariat. (C) Histogram depicting the effect of theophylline on the splicing of AdML 21AG and AdBPT pre-mRNAs. Splicing was calculated as the ratio of spliced mRNA to the total and normalized to the control (no theophylline). Data represent mean ± standard error of the mean (SEM) of three independent experiments. Asterisk represents significant change as compared to the control (*, P < 0.0005). (D) Theophylline inhibits the splicing of AdBPT15AG pre-mRNA in a dose-dependent manner. 32P-labeled AdBPT15AG pre-mRNA was incubated in HeLa nuclear extracts in the absence or with increasing concentration of theophylline for 2 h as described in (B). % Splicing calculated as described above and plotted against theophylline (mM). The values are expressed as mean ± SEM.

Mentions: To investigate whether theophylline-induced sequestering of BPS would allow conditional control of splicing, we constructed a series of AdML pre-mRNA derivatives in which BPS was embedded within high-affinity theophylline binding aptamer (TBA) (Fig. 1A and see Additional File 1). These pre-mRNAs differ in terms of the distance of aptamer sequence from the 3' ss AG. 32P-labeled pre-mRNAs were transcribed as runoff transcripts using T7 RNA polymerase and gel purified RNAs were incubated in HeLa nuclear extract under the conditions that support in vitro splicing. The results presented in Fig. 1B demonstrate that these substrates underwent normal splicing, albeit with lower efficiency compared with the parent pre-mRNA (Fig. 1B compare lane 2 with lanes 5, 7 and 9). Significantly, splicing reactions performed in the presence of theophylline gave rise to lower yields of spliced mRNA, suggesting that theophylline-mediated sequestering of branchpoint inhibits pre-mRNA splicing (Fig. 1B, compare lanes 5, 7 and 9 with lanes 6, 8 and 10, respectively). Quantitation of these data indicate that theophylline inhibited the splicing of AdBPT pre-mRNAs by ~65–90%, but had no effect on the splicing of AdML 21AG, a pre-mRNA that does not contain theophylline-binding aptamer (Fig. 1C). We chose AdBPT15AG for further experiments because of its better splicing efficiency in the absence of theophylline; while 17% of AdBPT15AG pre-mRNA was converted into mRNA, only ~7% and 9% of AdBPT12AG and AdBPT18AG pre-mRNAs gave rise to spliced product, respectively.


Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing.

Kim DS, Gusti V, Dery KJ, Gaur RK - BMC Mol. Biol. (2008)

Insertion of BPS within theophylline-responsive riboswitch confers ligand-dependent control of splicing. (A) Schematic diagram of AdBPT pre-mRNAs. Underlined sequence and encircled adenosine residue represents the BPS and branch nucleotide, respectively. Open boxes represent exon sequences and horizontal lines between exons indicate introns. Arrow specifies 5' and 3' splice sites. The boxed residues within the secondary structure of theophylline binding aptamer are conserved for theophylline binding. The numbering system follows according to the reference [29]. (B) In vitro splicing of AdML 21AG and AdBPT pre-mRNAs. 32P-labeled pre-mRNAs were incubated in HeLa nuclear extracts at 30°C for 2 h in the absence or presence of theophylline (for experimental details, see methods section). Total RNA isolated from each reaction was fractionated on a 13% polyacrylamide denaturing gel. The bands corresponding to pre-mRNA substrates, intermediates and spliced products are indicated on the right. M, Century™-plus RNA size marker (Ambion). Asterisk (*) indicates degraded lariat. (C) Histogram depicting the effect of theophylline on the splicing of AdML 21AG and AdBPT pre-mRNAs. Splicing was calculated as the ratio of spliced mRNA to the total and normalized to the control (no theophylline). Data represent mean ± standard error of the mean (SEM) of three independent experiments. Asterisk represents significant change as compared to the control (*, P < 0.0005). (D) Theophylline inhibits the splicing of AdBPT15AG pre-mRNA in a dose-dependent manner. 32P-labeled AdBPT15AG pre-mRNA was incubated in HeLa nuclear extracts in the absence or with increasing concentration of theophylline for 2 h as described in (B). % Splicing calculated as described above and plotted against theophylline (mM). The values are expressed as mean ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Insertion of BPS within theophylline-responsive riboswitch confers ligand-dependent control of splicing. (A) Schematic diagram of AdBPT pre-mRNAs. Underlined sequence and encircled adenosine residue represents the BPS and branch nucleotide, respectively. Open boxes represent exon sequences and horizontal lines between exons indicate introns. Arrow specifies 5' and 3' splice sites. The boxed residues within the secondary structure of theophylline binding aptamer are conserved for theophylline binding. The numbering system follows according to the reference [29]. (B) In vitro splicing of AdML 21AG and AdBPT pre-mRNAs. 32P-labeled pre-mRNAs were incubated in HeLa nuclear extracts at 30°C for 2 h in the absence or presence of theophylline (for experimental details, see methods section). Total RNA isolated from each reaction was fractionated on a 13% polyacrylamide denaturing gel. The bands corresponding to pre-mRNA substrates, intermediates and spliced products are indicated on the right. M, Century™-plus RNA size marker (Ambion). Asterisk (*) indicates degraded lariat. (C) Histogram depicting the effect of theophylline on the splicing of AdML 21AG and AdBPT pre-mRNAs. Splicing was calculated as the ratio of spliced mRNA to the total and normalized to the control (no theophylline). Data represent mean ± standard error of the mean (SEM) of three independent experiments. Asterisk represents significant change as compared to the control (*, P < 0.0005). (D) Theophylline inhibits the splicing of AdBPT15AG pre-mRNA in a dose-dependent manner. 32P-labeled AdBPT15AG pre-mRNA was incubated in HeLa nuclear extracts in the absence or with increasing concentration of theophylline for 2 h as described in (B). % Splicing calculated as described above and plotted against theophylline (mM). The values are expressed as mean ± SEM.
Mentions: To investigate whether theophylline-induced sequestering of BPS would allow conditional control of splicing, we constructed a series of AdML pre-mRNA derivatives in which BPS was embedded within high-affinity theophylline binding aptamer (TBA) (Fig. 1A and see Additional File 1). These pre-mRNAs differ in terms of the distance of aptamer sequence from the 3' ss AG. 32P-labeled pre-mRNAs were transcribed as runoff transcripts using T7 RNA polymerase and gel purified RNAs were incubated in HeLa nuclear extract under the conditions that support in vitro splicing. The results presented in Fig. 1B demonstrate that these substrates underwent normal splicing, albeit with lower efficiency compared with the parent pre-mRNA (Fig. 1B compare lane 2 with lanes 5, 7 and 9). Significantly, splicing reactions performed in the presence of theophylline gave rise to lower yields of spliced mRNA, suggesting that theophylline-mediated sequestering of branchpoint inhibits pre-mRNA splicing (Fig. 1B, compare lanes 5, 7 and 9 with lanes 6, 8 and 10, respectively). Quantitation of these data indicate that theophylline inhibited the splicing of AdBPT pre-mRNAs by ~65–90%, but had no effect on the splicing of AdML 21AG, a pre-mRNA that does not contain theophylline-binding aptamer (Fig. 1C). We chose AdBPT15AG for further experiments because of its better splicing efficiency in the absence of theophylline; while 17% of AdBPT15AG pre-mRNA was converted into mRNA, only ~7% and 9% of AdBPT12AG and AdBPT18AG pre-mRNAs gave rise to spliced product, respectively.

Bottom Line: Several lines of evidence suggest that theophylline-dependent inhibition of splicing is highly specific, and thermodynamic stability of RNA-theophylline complex as well as the location of BPS within this complex affects the efficiency of splicing inhibition.Finally, we have constructed an alternative splicing model pre-mRNA substrate in which theophylline caused exon skipping both in vitro and in vivo, suggesting that a small molecule-RNA interaction can modulate alternative splicing.These findings provide the ability to control splicing pattern at will and should have important implications for basic, biotechnological, and biomedical research.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA. dskim11@gmail.com

ABSTRACT

Background: Despite tremendous progress in understanding the mechanisms of constitutive and alternative splicing, an important and widespread step along the gene expression pathway, our ability to deliberately regulate gene expression at this step remains rudimentary. The present study was performed to investigate whether a theophylline-dependent "splice switch" that sequesters the branchpoint sequence (BPS) within RNA-theophylline complex can regulate alternative splicing.

Results: We constructed a series of pre-mRNAs in which the BPS was inserted within theophylline aptamer. We show that theophylline-induced sequestering of BPS inhibits pre-mRNA splicing both in vitro and in vivo in a dose-dependent manner. Several lines of evidence suggest that theophylline-dependent inhibition of splicing is highly specific, and thermodynamic stability of RNA-theophylline complex as well as the location of BPS within this complex affects the efficiency of splicing inhibition. Finally, we have constructed an alternative splicing model pre-mRNA substrate in which theophylline caused exon skipping both in vitro and in vivo, suggesting that a small molecule-RNA interaction can modulate alternative splicing.

Conclusion: These findings provide the ability to control splicing pattern at will and should have important implications for basic, biotechnological, and biomedical research.

Show MeSH
Related in: MedlinePlus